1. Field of the Invention
The invention concerns a process for culturing mammalian cells which require contact with cell surface proteins for activation, differentiation and/or proliferation as well as devices for culturing these cells.
2. Description of the Related Art
The culture of human cells is of major importance for various therapeutic approaches. Human cells cultured in vitro are for example required in adoptive immunotherapy with autologous or allogenic cells. Efficient processes for culturing haematopoietic progenitor and stem cells which for example can be transplanted into the patient after radiation therapy or chemotherapy are also of major importance.
Cytotoxic T lymphocytes (CTL) are responsible for eliminating pathogenically changed endogenous cells such as e.g. cells infected with viruses or tumour cells. In adoptive immunotherapy, lymphocytes of the patient are activated in vitro and then reimplanted. Such an activation can for example be carried out by adding interleukin 2 (IL2) to promiscuous killer cells (Thiele, D. et al., Immunology Today 10 (1989) 375-381). Such cells are then referred to as lymphokine-activated killer cells (LAK cells) (Rosenberg, Immunology Today 9 (1988) 58-62). However, LAK cells are also obtained in the stimulation with IL2 which are directed against healthy endogenous cells (Chen, B. et al., Cell Immunol. 118 (1989) 458-469). In a further method for adoptive immunotherapy, the lymphocytes to be activated are cultured in the presence of autologous tumour cells (Mixed Lymphocyte Tumor Cultures, Fossati, G. et al., International Journal of Cancer, 42 (1988) 239-245). A further method is described in WO 94/23014. According to this method lymphocytes are activated to form tumoricidal cells in a co-culture with a mammalian cell line while avoiding an allogenic stimulation. Fragments or vesicles of this cell line can also be used instead of the cell line described in this reference.
Suspended vital tumour cells or fragments thereof which have been previously advantageously inactivated by chemotherapy or radiotherapy are usually used for the ex vivo activation of CTLs. However, this process has major disadvantages. The inactivation of the tumour cells is complicated (irradiation, handling of toxic substances). It cannot be excluded that vital tumour cells or DNA from tumour cells are carried over into the transplant during transplantation.
Furthermore the inactivation of cells can lead to receptor modulations. Also the secretion of inhibitory molecules by for example inactivated tumour cells that are still alive cannot be excluded. Also the geometric/mechanical problem of the optimal cell density of effector to activator cells (probability of hits) is time-consuming and can only be determined empirically.
The immobilization of biological effectors on cell culture surfaces is used to activate cells and to proliferate them. Thus for example anti-T-cell antibodies are immobilized by preincubation on cell culture vessels by means of non-covalent binding. T cells that are added proliferate by binding/interaction of their CD3 receptors with immobilized (CD3) antibodies (Geppert, T. D., Lipsky, P. E., The Journal of Immunology 6, Vol. 138 (1987) 1660-1666).
Antigen-specific CTL's can be induced in a similar process by immobilizing MHC molecules alone (Walden, P., et al., Nature, Vol. 315 (1985) 327-329) or embedded in synthetic, planar membranes (Watts, T. H. et al., Proc. Natl. Acad. Sci. USA, Vol. 81 (1984) 7564-7568). Moreover the T cell activation can be modulated by interactions with immobilized accessory molecules (Moy, V. T., Brian, A. A., J. Exp. Med. 175 (1992) 1-7).
Furthermore cells can be immobilized by non-covalent binding on cell culture vessel surfaces. The binding of monocytes on FCS-coated culture vessels and pulsing with specific antigens leads, after co-culture with peripheral blood lymphocytes, to an improvement of the antigen presentation with increased antibody production of the B cells (Jahn, S., et al., Allerg. Immunol. 33 (1987) 239-244).
The preparation of artificial lipid vesicles is state of the art. These liposomes can, on the one hand, be loaded with proteins embedded in the lipid membrane to improve cell targeting in vitro (Herrmann, S. H., Mescher, M. F., Proc. Natl. Acad. Sci. USA, Vol. 78, No. 4 (1981) 2488-2492; Bloemen, P. G. M., et al., FEBS Letters 357 (1995) 140-144; Bergers, J. J., et al., Journal of Controlled Release 29 (1994) 317-327; Gregoriadis, G., Immunology Today, Vol. 11, No. 3 (1990) 89-97). On the other hand, chemotherapeutic agents can be enclosed in such vesicles to increase the local dose in order to induce cell death (Brown, P. M., Silvius, J. R., Biochimica et Biophysica Acta 1023 (1990) 341-351). The in vivo administration of artificial anti-tumour vesicles (liposomes) for antigen immunotherapy also corresponds to the state of the art (Phillips, N. C., et al., Liposomes in the Therapy of Infectious Diseases and Cancer, 1989, Alan R. Liss, Inc. (ed.), p. 15-24; Bergers, J. J., et al., Cancer Immunol. Immunother. 34 (1992) 233-240; Papahadjopoulos, D., Gabizon, A., Annals of the New York Academy of Sciences, Vol. 507, R. L. Juliane (ed.), 1987, 64-74).
In the previously known processes artificial liposomes/vesicles are either used in solution by non-covalent binding to carriers or in vivo. It is also known that cells labelled covalently via a binding partner can be used in a cell separation process to enable separation of bound specific cells from undesired cells by means of immobilized antibodies (EP-A 0 701 130).
A process for culturing haematopoietic progenitor stem cells using feeder layers of stroma cells is described in WO 95/02040. However, the preparation of such feeder layers is time-consuming.